Biotinylated dextran amine

Five adult WT and five Dcc^+/− animals were anesthetized with ketamine/xylazine (100/10 mg/kg body weight) and a hand drill (Dremel) was used to create a small opening in the skull; 5 μl of biotinylated dextran amine (10% in PBS, Invitrogen, 10,000 MW) was injected unilaterally into the motor cortex with a syringe (Hamilton, 80 300) and the animal was sutured and allowed to recover. After 14 d, animals were perfused in 4% paraformaldehyde (PFA) in PBS and the spinal cord and brain were dissected and postfixed in 4% PFA overnight before cryoprotection in 30% sucrose in PBS and freezing of segments in tissue freezing medium (O.C.T. compound); 30-μm cryosections of brain and spinal cord were incubated in streptavidin-488 (Jackson ImmunoResearch, 1:200 in PBS + 0.1% Triton X-100) for 2 h at room temperature, mounted, and imaged with a Leica DM4000 fluorescent microscope.

Mice were anesthetized by intraperitoneal injection of a ketamine/xylazine (86 mg·kg⁻¹/13 mg·kg⁻¹, IP) mixture and secured in a headholder. Analgesia was achieved by buprenorphine (5 mg·kg⁻¹, SC). Callosal connections were labeled by 30–40 pressure injections (20 nl each) of the retrograde tracer bisbenzimide (BB, 5% in H₂O, Sigma) into the right occipital cortex. Interareal projections were labeled by iontophoretic injections (3 µA, 7 s on/off duty cycle for 7 min) of the anterograde tracer biotinylated dextran amine (BDA; 10,000 molcular weight, 5% in H₂O; Invitrogen) using a coordinate system whose origin was the intersection between the midline and a perpendicular line drawn from the anterior border of the transverse sinus at the posterior pole of the occipital cortex. The coordinates of the injected areas were (anterior/lateral in mm): V1 (1.1/2.6); LM (1.4/4.1); PM (1.9/1.6). Mice were randomly assigned for injections of a particular area.